Magnetic resonance system and method to detect and confirm analytes

ABSTRACT

A system and method are provided to detect target analytes based on magnetic resonance measurements. Magnetic structures produce distinct magnetic field regions having a size comparable to the analyte. When the analyte is bound in those regions, magnetic resonance signals from the sample are changed, leading to detection of the analyte.

RELATED APPLICATIONS

This application is a continuation application of U.S. patentapplication Ser. No. 11/335,995 entitled A MAGNETIC RESONANCE SYSTEM ANDMETHOD TO DETECT AND CONFIRM ANALYTES filed Jan. 20, 2006 (now U.S. Pat.No. 7,781,228, issued Aug. 24, 2010), which claims the benefit of U.S.provisional application Ser. No. 60/673,382, filed Apr. 21, 2005, titledBIO-HAZARD SUBSTANCE DETECTOR AND IDENTIFIER, and Ser. No. 60/669,019,filed Apr. 7, 2005, titled SHIPPING CONTAINER INSPECTION DEVICE. Thisapplication claims the benefit of the aforementioned patentapplications, which are incorporated herein by reference in theirentirety.

GOVERNMENT INTEREST

This invention was made with U.S. Government support under one or moreof the following contracts: Naval Air Warfare Center n68335-02-c-3120,Department of Homeland Security contracts NBCHC060017 andHSHQPA-05-9-0039. The U.S. Government has certain rights in thisinvention.

BACKGROUND

1. Field of the Invention

The present invention generally relates to the field of analytedetection and additionally relates to detecting analytes using magneticresonance.

2. Related Art

Detection technology for specific analytes spans a wide range oflaboratory instrumentation and techniques including liquid and gaschromatography (LC and GC, respectively), mass spectrometry (MS),nuclear magnetic resonance (NMR) spectroscopy, polymerase chain reaction(PCR), optical spectroscopy and fluoroscopy, Fourier transform infrared(FTIR) spectroscopy, and ion mobility instruments. Today's chemicalanalysis instruments however, are large and expensive, require a skilledoperator, involve complex sample preparation, and require substantialamounts of time for analysis.

There is a critical need worldwide for improved detection of specificchemicals and microbes. For example, in the area of national security, asystem is needed to detect biological agents, toxins, and chemicalweapons to provide early alert in case of a terrorist attack. Such adetection capability could also be used to search for clandestine siteswhere such weapons are under development or in production, thus enablingaction to prevent their use. A system is also needed to scan mail andpackages to detect a terrorist attack.

Improved pathogen detection is also needed for medical science.Sensitive detection of DNA or proteins associated with avian flu, bovinespongiform encephalopathy (more commonly referred to as “mad-cowdisease”), or severe acute respiratory syndrome (SARS) would enableintervention to avoid a pandemic. Broad clinical use of such a systemwould assist in identifying ordinary diseases or serious illnesses,greatly assisting physicians in diagnosis.

Detection of various chemicals is also needed for industrialapplications to detect toxic industrial chemicals (TICs) and toxicindustrial materials (TIMs). Such a system would enable leak detection,process control, detection of material degradation, control ofconcentration, and a host of other process applications in a wide rangeof industries.

Improved detection is also needed in agriculture and food production, aswell as a means to detect contamination, spoiling, or poisoning of food.Food includes for example, items such as drinking water and fruitjuices. There is also a need in forensic testing, including for example,searching for specific DNA sequences in a sample at the search site.

Magnetic resonance detection techniques are under development involvingnanometer-scale paramagnetic particles (nanoparticles) which havepreviously been used as MRI contrast agents. The particles comprise acore of paramagnetic or superparamagnetic (both generally referred toherein as paramagnetic) material, coated by a shell of nonmagneticmaterial which are adorned with reactant molecules to promote binding totarget cells such as pathogens, tumor cells, etc. Nanoparticles areinjected into a patient prior to MRI analysis. They bind to the targetcells, cause a local change in the MRI image properties, and enabledetection or localization of the target cells.

The nanoparticles have also been used in vitro. Dissolved or suspendedin a liquid medium, the nanoparticles bind to target cells or moleculesin the medium. The nanoparticles and analytes may form aggregatesincorporating dozens to thousands of nanoparticles. Such aggregates aredetectable by light scattering, atomic-force microscopy, electronmicroscopy, and in some cases by NMR effects. See, for example, U.S.Pat. No. 5,254,460 to Josephson et al.

Target-specific reactants can be mounted onto the nanoparticles toprovide analyte-specific selectivity. A disadvantage is the need to formaggregations comprising a plurality of nanoparticles and a plurality oftarget cells or molecules, because aggregation occurs only when eachnanoparticle is bound to multiple analytes, and each analyte is bound tomultiple nanoparticles. Aggregation can be inhibited by geometricaleffects such as a variation in size among nanoparticles. Substantialtime may be required for the aggregations to form.

Prior studies on agglomeration were conducted on benchtop relaxometersand high-field MR instruments. Sample preparation and insertion into theNMR tube is tedious. Hand mixing of each sample and then insertion ofsample is time consuming and miss the important binding event that takesplace early on when an analyte binds to the nanoparticle. A compact andautomated instrument is required to speed up measurements. Also, it isimportant to understand the phenomena describing the changes seen in themeasurement from a basic physics and biochemistry standpoint.

Earlier studies did not model the change in T2 effects from a physicsstandpoint. Simple agglomeration effects were observed through opticalmeans (microscopes) to establish the phenomena relating change in T2. Inaddition, early studies did not take advantage of stoichiometry controlof the nanoparticles to adapt the measured parameters for variousapplications leading to specific NMR products.

Earlier studies used samples that were pure and not subject tointerferences such as dust, acids, etc. Moreover, there was norequirement for fast measurements combined with no interference fromclutter and near neighbor molecules, cost of overall system, low falsealarms and high probability of detection. There was also no definedrange of analyte concentrations to be detected.

Earlier studies did not consider use of improved paramagnetic materialssuch as compounds of iron, cobalt and nickel leading to strongermagnetization and improved sensitivity.

SUMMARY

A system and method are provided which can detect target analytes basedon magnetic resonance measurements. In one aspect analytes are detectedusing specific nanoparticles in the form of magnetic resonancenanoswitches.

In one aspect of the invention, systems and methods detect targetedanalytes with very high specificity, despite near-neighbor interference,dirt, clutter, biological interferents such as mold spores,proteinaceous interferents such as skim milk and ova albumin,paramagnetic interferents such as hemoglobin and humic acid (containingchelated iron), environmental interferents such as the so-called Arizonadust, diesel soot, etc.

One aspect of the invention includes a system and method for detectinganalytes in a liquid medium. In another aspect analytes may beintroduced as aerosol, hydrosol, and in complex media such as food.

The system includes a magnetic resonance system to detect resonancesignals from the liquid, a magnetic field passing through that liquid,and a region within the liquid where the magnetic field has a distinctproperty such as a particular value or gradient. Liquid within thatregion produces magnetic resonance signals which depend on the fieldproperty, and liquid outside that region may also be influenced by theregion due to diffusion. A material having particular affinity for theanalyte is adjacent to the region. The analyte binds to or is held bythe affinity material and displaces liquid from that region, thusaltering the magnetic resonance signals and revealing the analyte.

A system for detecting an analyte comprises: a sample which contains theanalyte within a liquid medium, means for generating a first magneticfield within the liquid, means for generating a second magnetic fieldwithin a special region within the liquid, means for holding the analytewithin the special region, a magnetic resonance instrument capable ofmeasuring magnetic resonance signals from the liquid, and means foranalyzing those signals to determine whether the liquid occupies thespecial region. Alternatively, liquid passing through the special regioninfluences the magnetic resonance signals of the remaining liquid. Thesecond magnetic field is distinct from the first magnetic field so thatthe signals of the liquid residing within the special region differdetectably from signals of the liquid located exterior to the specialregion. When present, an analyte displaces liquid from the specialregion. Thus if the signals show that liquid occupies the specialregion, analyte must be absent. If the signals show that the liquid isdisplaced from the special region, then the analyte must be present, andis thus detected.

The analyte can be any molecule, molecular complex, microbe, chemical,or material which can be contained in the liquid medium, and whichdisplaces the liquid when so contained. Examples of analytes includebio-molecules such as proteins, DNA, RNA, or fragments or complexesthereof; enzymes, small molecules, organisms, microbes such as whole ordisrupted viruses or bacteria; whole or disrupted cells from otherspecies including humans, non-biological chemicals such as chemicalweapon molecules, explosives, insecticides, pharmaceuticals, andindustrial chemicals.

In one embodiment the liquid contains the analyte. Here “contains” meansthat the analyte is dissolved, suspended, emulsified, or otherwisewholly enclosed in and dispersed within the liquid. Also, the analytedisplaces the liquid, meaning that molecules of the analyte can notco-occupy space with molecules of the liquid.

The liquid can be any fluid material that includes a nucleus havingnon-zero spin. Only nuclei with non-zero spin give rise to the NMRphenomena. The liquid includes such nuclei when molecules comprising theliquid comprise a nucleus with non-zero spin, such as hydrogen in thewater molecule. Alternatively, the liquid may include such nuclei assolutes or suspensions, such as a fluoridated solute which generatesmagnetic resonance signals at the ¹⁹F Larmor frequency.

In a further aspect a system includes a first magnetic field whichpasses through the liquid. The first magnetic field may be produced byan electromagnet, permanent magnet, superconducting coil, or any othersource. Normally the first magnetic field is a static and substantiallyuniform magnetic field that can be in the range of 0.01 Tesla to 20Tesla, and is a part of the magnetic resonance system.

A second magnetic field is generated in a special region of the sample.The second magnetic field is distinct from the first magnetic field insome parameter that is detectable using magnetic resonance. For example,the second magnetic field may differ from the first magnetic field inmagnitude, orientation, uniformity, gradient, or any other detectableparameter. A second magnetic field generator or means for generating thesecond magnetic field may be a nanoparticle, suspended in the liquid andimmersed in the first magnetic field or applied field. In one embodimentthe nanoparticle becomes magnetized and produces a dipole-shaped fieldthat adds vectorially to the applied field, producing a net magneticfield. The special region is that volume occupied by the distinctmagnetic field. When the distinct magnetic field is caused by ananoparticle the special region is that nanometer-scale volume adjacentto but exterior to the surface of the nanoparticle, where the net fielddiffers substantially from the applied magnetic field. Alternatively,the special magnetic field region could be produced by paramagnetic ionssuch as chelated iron or gadolinium instead of nanoparticles. Anadvantage of this approach is that diffusion-limited reaction rates maybe increased due to the higher mobility of metal-ion chelates. Similarions are used in MRI (Gd-DTPA and Gd-DOTA).

Alternatively, the special magnetic field region is produced byparticles or structures having a size larger than nanometer-scale,provided that the magnetic resonance signals differ detectably whenanalyte is present or absent. For example, shaped magnetic structuresmay provide two specific values of the magnetic field in two regions,and the analyte binding molecules could be coupled to only one of thefield regions. The detection measurement is then a spectral analysis ofthe composite magnetic resonance signal, which will exhibit twofrequency peaks corresponding to the two field regions when no analyteis present, or only a single peak when analyte obscures one of the fieldregions.

In one aspect, temperature cycling is used to accelerate binding betweenthe analyte and nanoparticle. This shortens the binding event time byincreasing the mobility of the analyte and/or the nanoparticle. When anenergy barrier inhibits binding, higher temperatures improve the rate ofbinding. Temperature cycling may include heating and cooling or viceversa. Then the sample is measured in the magnetic resonance instrument.

In one aspect, the system includes a mechanism or binding agent forholding the analyte in the special region, to displace the liquid fromthe special region, leading to detection of the analyte. Such a bindingagent can include any material surface or molecule for which the analytehas an affinity. Such holding may be accomplished by hydrogen bonds,ionic forces, covalent bonds, sulfide bridges, van der Waals forces,electrostatic forces, or any other type of molecular or materialattachment or affinity ligand. The binding agent is positioned adjacentto the region of shaped magnetic field so that the target molecule, whenbound, occupies that region and excludes the liquid therefrom. Forexample, the binding agent may be an antibody raised against an analyteprotein, or DNA complementary to analyte DNA sequences. Preferably thebinding agent also has null affinity or negative affinity for allsolutes other than the analyte that may be present. In addition to DNA,other holding means can be used such as aptamers, small molecules, etc.Targets include, but are not limited to the following:

-   -   a. An antibody that recognize and binds to an antigen    -   b. an oligonucleotide or DNA sequence complementary to a DNA- or        RNA-target    -   c. a DNA- or RNA-aptamer that binds to a target protein,        bacteria, virus, yeast or fungus.    -   d. a protein or peptide that binds to a target protein,        bacteria, virus, yeast or fungus.    -   e. a pseudopeptide composed of unnatural amino acids with a        stronger binding to a target or better environmental stability.    -   f. a small molecule or combination of small molecules that can        bind to a target.    -   g. monosaccharides, polysaccharides, carbohydrates and sugars        that can bind to a target protein, bacteria, virus, yeast or        fungus.

A further aspect includes a magnetic resonance instrument, which iscapable of exciting and detecting magnetic resonance signals from theliquid medium. Existing magnetic resonance systems may perform thisfunction. More preferably, the instrument is a simple, compact,automated, single-purpose magnetic resonance system which can performthe detection measurement automatically. The system measures signalsrelated to the presence or absence of liquid, affected by the secondmagnetic field in the special region. For example, when the magnitude ofmagnetic field in the special region differs from that in the rest ofthe liquid, then the magnetic resonance system can measure the spectralcontent of the magnetic resonance signals to determine the magneticfield from which the signals emerged. Thus by analyzing for the Larmorfrequency of the liquid in the special region, the system determineswhether liquid occupies that region.

An alternative measurement is the spin-spin relaxation time (T2) of theliquid. T2 is affected when the magnetic field in the special region hasstrong gradients, and particularly when the liquid diffuses throughthose gradient fields in times short compared to the measurement. Thusthe system can determine the presence of analyte by measuring the T2 ofthe liquid to determine if depolarization is occurring in the specialregion.

In one aspect, the compact magnetic resonance system can measure eithera positive or negative change in T2. Agglomeration is described in theJosephson patent as the formation of a large supermolecular assembly ofmolecules. Agglomeration necessarily occurs later in time than thebinding event. In the case of agglomeration, all measurements show anegative T2 change. Likewise, the parameter defined as “positive 1/T2”in Josephson represents a negative change in T2. Agglomeration isdescribed by Josephson as a process where several molecules attach toeach other and they form assemblies large enough to change the T2 of thewater. In one embodiment, the inventive system measures T2 changes dueto the analyte binding event, leading to positive and negative T2changes prior to agglomeration.

In one aspect, the compact magnetic resonance system measures baselineT2 on the nanoparticles or nanoparticle solution first, which is thenmixed with the analyte and T2 is measured again to determine whether achange in T2 has occurred. This is done to ensure the correctconcentration of nanoparticles and consistent stoichiometry, as well asto cancel out metering and mixing errors, variations in nanoparticleproperties, fluidic transport errors, etc.

In one embodiment the inventive system can detect analyte by measuringmagnetic resonance signals from the sample at a single time.Alternatively, the system can perform a series of measurements spanninga period of time and can compare or analyze the measurements to improvethe detection of analyte. For example, the binding between analyte andnanoparticles may proceed during an interval which is longer than thetime required for a particular measurement. Then the system can performthe measurements repeatedly to observe the changes caused by thebinding. As another example, the analyte may first bind to nanoparticlesto form binaries, causing a positive shift in T2, and thereafter thebinaries may combine to form agglomerates, causing a negative shift inT2. Such data can greatly enhance the quality of the result by reducingthe false alarm rate, providing a lower detection threshold, andenhancing the detection probability for a given quantity of analyte.

The inventive system can derive parameters related to reaction kineticsfrom repeated measurements on the same sample, including a rate ofchange of a parameter or an accumulated reaction parameter. Theseresults can then be used to guide additional measurements to confirm orclear the initial indication. For example, if a sample exhibits a smallbut suspicious T2 change soon after mixing, the system can initiate aseries of tests to determine the rate of change in T2 over a period oftime. Then, if those later results confirm that the analyte is present,an alarm can be issued, but if the follow-on measurements indicate noanalyte, then the initial suspicion may be cleared, thereby averting afalse alarm. Using a provisional re-scan protocol, combined with arate-magnitude analysis, the system enhances both reliability andthreshold sensitivity.

Based on experimental results and theoretical modeling, a positive T2change is due to analyte displacing water molecules upon binding to thenanoparticles, and negative T2 change is due to repeated dephasing ofwater molecules within a cage structure formed by multiplenanoparticles. In addition, the positive or negative T2 change can bepromoted by processing and stoichiometry. For example, the ratios ofnanoparticles and the reagent can be adjusted to provide negative orpositive T2 changes. In some circumstances it can be important tomeasure both negative and positive T2 effects to eliminate interferencefrom the base sample. For example, a test sample contaminated with aparamagnetic ion, such as humic acid with chelated iron, causes areduction in the T2 of the mixture. Thus if there is an analyte mixed inwith the humic acid, it can be measured in the following manner. First,measure the sample prior to mixing the nanoparticles, to generatebaseline T2 measurement. Then, mix the nanoparticles and measure thechange in T2, positive or negative, to reveal the analyte.Alternatively, in the event that a separate baseline measurement cannotbe taken, it is useful to mix nanoparticles which lead to a positive T2difference upon binding to the analyte, so that the analyte may bedetected despite the contaminant.

“Nanoparticle multiplexed mixtures” are nanoparticle preparationssensitized to multiple analytes. There are two multiplexing scenarios.First, each nanoparticle in the mixture is sensitized to a specificanalyte. Second, a single nanoparticle is sensitized to multipleanalytes.

In one embodiment, an automated air monitoring system includes inlets toadmit the sample along with air, a collector that gathers the samplematerial and concentrates it into a liquid form, called a raw sample,and a fluidics system. The fluidics system holds the raw sample, forexample in a container and provides consistent metering of the rawsample, for example via an outlet tube using a pump such as aperistaltic pump. Metered sample is mixed with selected nanoparticleswhich may be in water, for example, drawn from reservoirs via an outletby a pump. As soon as the reaction takes place the fluidics system movesthe sample into the sample area of the magnetic resonance system formeasurement, for example, via a tube driven by a pump. Alternatively,sample mixing and processing may take place within the magneticresonance system. The fluidics system may include means for cell lysingwherein the fluidics system may lyse or disrupt cells or viruses in thesample to release proteins, RNA, or DNA of the target cell. The fluidicssystem may also have a temperature control built in to speed up thebinding event. Fluidics system also may have an overall system cleaningsolvent to eliminate contamination. The cleaning solvent or rinsingagent can be drawn from a reservoir and pumped through the tube whichdelivers the samples to the sample area. The fluidics system also allowspositive and negative control tests to ensure the overall system isfunctional, and performs calibration tests using calibration standards.

In one embodiment, chelates are used in place of nanoparticles togenerate the distinct magnetic field region and to bind to analyte. Anadvantage of using chelated ions is that it allows faster diffusionthrough the liquid medium to speed up diffusion-limited processes. Onthe other hand, with nanoparticles one can tailor the affinity moleculesto select the analyte desired, whereas chelates occur only in specificmolecular forms. Nanoparticles have more area to attach the affinitymolecules compared to chelates. Nanoparticles can be decorated withchelates for binding to analytes, explosives and chemicals.

In one aspect, the second magnetic field region, being generated byparamagnetic cores or chelates or other magnetic structures, has a sizecomparable to the size of the analyte, so that the bound analyte justfills the second magnetic field region, excluding the liquid from thatregion, thus providing highest signal and highest sensitivity. Forexample, when the analyte is a relatively small molecule such as anexplosive vapor molecule or a chemical weapon molecule, then the size ofthe second magnetic field region is preferably chosen to be in the rangeof 1 to 10 nm. To detect a larger analyte, such as a toxin or DNA orvirus particle, then the size of the second magnetic field region wouldbe 10 to 100 nm. When the analyte is an even larger objects such as abacterium, the size of the second magnetic field region may be 100 to1000 nm or larger as needed to match the analyte.

The nanoparticles may include structures that provide an opticalsignature. For example, fluorescent dyes or centers may be attached toor included within the nanoparticles, and may be exposed to photons ofsufficient energy to excite fluorescence, causing emission offluorescence photons having an energy different from, and usually lowerthan, the excitation photons. The excitation and fluorescence photonsmay be in the ultraviolet, visible, or infrared range. Detection of thefluorescence photons provides a measure of the nanoparticleconcentration. In addition, the structures may be modified when analytebinds to the nanoparticle, and such action may result in a detectablechange in the fluorescence such as a change in the intensity or energyof the fluorescence photons. Detection of this change would provide anindication, independent of magnetic resonance measurements, that analytebinding has occurred and thus that analyte is present in the sample.

Other features and advantages of the invention will be apparent from thefollowing detailed description, the claims and the appended drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

The details of the present invention, both as to its structure andoperation, may be gleaned in part by study of the accompanying drawings,in which like reference numerals refer to like parts, and in which:

FIG. 1 is a schematic representation of a nanoparticle showing theapplied magnetic field and the second magnetic field around thenanoparticle.

FIG. 2 is a graph of the net magnetic field surrounding the nanoparticleof FIG. 1.

FIG. 3 is a plot of the magnitude of the magnetic field gradient aroundthe nanoparticle.

FIG. 4 is a plot of the field gradient magnitude along the axis of theparticle.

FIG. 5 which is a functional block diagram of a magnetic resonancesystem.

FIGS. 6a-d is a representation of four configurations of the antenna.

FIG. 7 is a schematic representation of one embodiment of a magnet.

FIG. 8 is a circuit diagram of a buffered oscillator.

FIG. 9 is a schematic illustration of an installation having onecontroller and multiple sensor units.

FIG. 10 shows a schematic of an HVAC monitor system.

FIGS. 11a-e depict an embodiment of a fixed installation system andthree collector intakes.

FIG. 12 is a front perspective view of a hand-portable system.

FIG. 13 is a block diagram of a system adapted to a medical diagnosticapplication.

DETAILED DESCRIPTION

After reading this description it will become apparent to one skilled inthe art how to implement the invention in various alternativeembodiments and alternative applications. However, although variousembodiments of the present invention will be described herein, it isunderstood that these embodiments are presented by way of example only,and not limitation. As such, this detailed description of variousalternative embodiments should not be construed to limit the scope orbreadth of the present invention as set forth in the appended claims.

Magnetic Resonance

A brief summary of the technical elements used in certain embodiments isprovided herein. The analyte or target molecule is contained in amedium, preferably a liquid such as water, which includes an atomicnucleus that has a non-zero spin, such as hydrogen. As is well known,(see for example, Pulse Methods in 1D & 2D Liquid-Phase NMR, Wallace S.Brey, Academic Press 1988), that the magnetic component of such anucleus becomes polarized or spatially oriented in a magnetic field, andmay be induced into magnetic resonance precession at a frequency givenby:f _(Larmor) =γB/2π,where B is the magnetic field strength at the position of the nucleus, γis the magnetogyric ratio of the nucleus, and f_(Larmor) is theresonance frequency or Larmor frequency (γ=2.675×10⁸ Tesla⁻¹ sec⁻¹ forthe hydrogen nucleus). The magnetic components, or magnetic moments, ofthe nuclei are vector quantities and add to give a resultant bulkmagnetization vector that is the NMR signal measured by NMRspectrometers.

Following a perturbation such as that employed in recording NMR signals(see below), the bulk magnetization vector recovers to its originalsteady state over time; this process is referred to as nuclear magneticrelaxation. Two fundamental time constants are used to describe thisrelaxation in terms of a single-exponential process. Recovery of thebulk magnetization along the direction of the first magnetic field isdescribed by the spin-lattice relaxation time or longitudinal relaxationtime, designated as T1. Typically, T1 is of order milliseconds toseconds. The single-exponential decay of bulk magnetization in the planeperpendicular to the direction of the first magnetic field is describedby the spin-spin relaxation time, or transverse relaxation time,designated as T2. For liquid signals, T2 is generally in the range of100 milliseconds or more. Solid samples on the other hand, generallyhave T2 values in the range of 1 to 100 microseconds.

A magnetic resonance measurement is performed by applying one or more RF(radio frequency) energy pulses to the sample and measuring the bulkmagnetization that becomes reoriented by the pulse. The RF pulses have afrequency equal to the Larmor frequency, and duration sufficient tocause the bulk magnetization vector to reorient into a planeperpendicular to the first magnetic field, where the bulk magnetizationvector (the NMR signal) can be recorded over time. The RF pulsestherefore, are usually multiples of 90 degrees.

Spin-spin relaxation is typically measured by a series of RF pulses togive rise to spin echo signals. A spin echo is generated by a 90-degreepulse followed by a small delay time (typically designated as τ),followed by a 180-degree pulse)(90°-τ-180°). A second τ, identical intime to the first, is used before the bulk magnetization vector isrecorded. The series of RF pulses and time delays is used to firstdephase the nuclear magnetic moments comprising the bulk magnetizationin the plane perpendicular to the first magnetic field during the firstτ, and refocus the remaining bulk magnetization in this plane during thesecond τ. This latter refocusing creates an echo signal, which can berecorded. The most common method to measure spin-spin relaxation is thatoriginally described by Can and Purcell (Carr, H. Y. and Purcell, E. M.:Effects of Diffusion on Free Precession in Nuclear Magnetic ResonanceExperiments, Physical Review 94, no. 3 (1954): 630-638), a modificationof the method described earlier by Meiboom and Gill (Meiboom, S. andGill, D.: Modified Spin-Echo Method for Measuring Nuclear RelaxationTimes, The Review of Scientific Instruments 29, no. 8 (1958): 688-691).The Carr-Purcell modified Meiboom-Gill (CPMG) method uses a series ofsmall time delays followed by 180-degree pulses after the initial90°-τ-180° sequence described above. This in turn is followed by theresultant bulk magnetization vector [90_(x)°-(τ-180_(y)°-τ-record)_(n)].The amplitudes of the spin echo signals are proportional to the bulkmagnetization remaining at the time of the echo, which becomessuccessively smaller as the number of sequences increases (as the valueof n increases). Therefore, measuring the amplitude of the bulkmagnetization vector after various values of n and fitting the data to asingle exponential decay with T2 as the relaxation time provides adirect measure of T2.

Paramagnetic Nanoparticle Fields

In a preferred embodiment, nanoparticles are employed to influence themagnetic field in a region close to the nanoparticles. The paramagneticor superparamagnetic core of the nanoparticle becomes magnetized when anexternal magnetic field is applied to it. Superparamagnetism is relatedto ferromagnetism in which the size of the magnetized body is too smallto form a magnetic domain. The superparamagnetic core exhibits a highpermeability and fairly high saturation field comparable to iron, butlittle or no hysteresis (H_(c)˜0). When placed in a magnetic field, thecore becomes strongly magnetized parallel to the direction of theapplied field. When the external field is removed, the core losesessentially all of its magnetization. Disregarding anisotropy and shapeeffects, the induced magnetic moment of the core is given by:m _(core)=(4π/3)(r _(core) ³)(χB ₀)where m_(core) is the dipole moment of the core, r_(core) is its radius,B₀ is the applied field, and χ is the susceptibility. Normally χ≈0 fornonmagnetic materials, χ>≈1 for superparamagnetic materials when B₀ isbelow a saturation field, and 1≦χ≦0 for B₀ above saturation. Forexample, magnetite (Fe₃O₄) is superparamagnetic with a susceptibility ofabout 1 for fields below saturation of about 0.5 Tesla.

The magnetized core produces a magnetic field which usually approximatesa dipole field, or the magnetic field produced by an ideal magneticdipole located at the center of the paramagnetic core of thenanoparticle. At locations outside the nanoparticle core, the dipolemagnetic field is parameterized as follows:B _(r)=2m _(core) cos θ/r ³B _(θ) =−m _(core) sin θ/r ³

Here B_(r) is the radial component of the dipole field, B_(θ) is thecircumferential component, r is the distance from the center of thecore, θ is the polar angle relative to the applied field, and m_(core)is the dipole moment.

The dipole field adds linearly to the applied field (as vectors),resulting in the net magnetic field. The Larmor frequency is determinedby the net magnetic field experienced by the polarized nucleus.Components of the dipole field orthogonal to the applied field causeprimarily a field rotation, whereas the dipole components parallel tothe applied field directly change the magnitude of the net field andtherefore change the Larmor frequency, relative to the undistortedapplied field. The net field B_(net), disregarding second order terms,and for r>>r_(core), is as follows:B _(net) =B ₀(1+4π/3(r _(core) /r)³χ(2 cos²θ−sin²θ))

In some embodiments the magnitude of the gradient of the net magneticfield is also important. The field gradient is given by:∇B _(net) =B ₀χ8π(r _(core) ³ /r ⁴)(−{r} cos²θ+{θ} cos θ sin θ)where curly brackets denote unit vectors in the r or θ directions.

Diffusion in a Liquid

Some embodiments include a liquid medium. The liquid contains theanalyte and the nucleus that emits the magnetic resonance signals. Thosesignals are influenced by diffusion, particularly the diffusion of themolecules of the liquid through the liquid, or molecular self-diffusion.Diffusion is formulated as follows:σ_(walk)=(2D _(molec) t)^(1/2)where σ_(walk) is the average distance traveled in an isotropicthree-dimensional random walk in time t, and D_(molec) is thetranslational diffusion coefficient. For example, D_(molec)=1.5×10⁻⁹m²/s for water at room temperature.

Magnetic resonance measurements are also influenced by spin diffusion, aphenomenon in which the spin or polarization of a nucleus isinterchanged with that of a nearby nucleus of the same type. Spindiffusion can distribute spin-dependent effects, such as depolarization,throughout the sample. For example, if a small fraction of the hydrogennuclei in water experience a depolarizing force, spin diffusion cancause all of the hydrogen in the sample to assume an averagedpolarization value.

A Model

This model addresses spin-dependent interactions between nanoparticlesand solvent, and provides a useful framework for quantifying theobserved T2 effects. It is used in some embodiments as the basis formeasuring and detecting analytes. A simplified nanoparticle is assumedto consist of a spherical core of superparamagnetic material, surroundedby a spherical shell of non-magnetic material, all in water. However,the model can be applied or modified for use with nanoparticles of othershapes and for use with other solvents. The model suggests the followingmechanisms for the observed T2 changes:

(1) Nanoparticles in solution reduce T2 relative to plain water. Themodel suggests that depolarization is due to a dipole magnetic fieldproduced by the magnetized core. The field distortion causes spins toprocess at different frequencies, leading to destructive interference.Although CPMG normally refocuses static field-nonuniformity effects, theBrownian motion of the water molecules causes them to enter and exit thefield distortions in a time shorter than the echo interval, therebymaking the spin dispersion time-dependent and breaking the CPMGrefocusing effect.

(2) When nanoparticles react with analyte, but do not agglomerate, theT2 increases. This may be due to the analyte molecules occupying part ofthe distorted-field region around the nanoparticle, thereby excludingwater from that region, thus reducing the spin dispersion and increasingT2.

(3) T2 decreases when nanoparticles and analyte agglomerate. This may bedue to the formation of a water-filled cage-like structure in whichwater molecules undergo repeated spin-dispersion collisions with thesurrounding nanoparticles. Sufficient repetition of incrementaldepolarization would reduce T2, despite the analyte occluding portionsof the non-uniform field regions.

(4) A single exponential usually fits the polarization decay curve. Thisis despite the fact that hydrogens close to nanoparticles are stronglydephased, while the general solvent sees only a uniform field, atwo-population system. However, the spin populations are rapidlyequilibrated across the sample by spin diffusion via homonuclearflip-flop interactions, resulting in a single averaged T2.

The model nanoparticle is depicted in cross section in FIG. 1 in thepresence of an applied magnetic field indicated by arrow 110. Thenanoparticle consists of a magnetizable core 112 which preferably isparamagnetic and more preferably is superparamagnetic. The induced localdipole-shaped field of the nanoparticle is represented by the dashedlines. The radius of the core should be large enough to produce asignificant magnetic field distortion in a large enough region toproduce a change in T2 of the liquid in that region. The radius of thecore should be small enough that the core does not become ferromagnetic.Typically the core radius is about 1 to 20 nm. Desirable properties ofthe core include high susceptibility at the applied magnetic fieldstrength, high saturation field preferably in excess of the appliedmagnetic field strength, chemical compatibility with the liquid medium,and very low remnant field. The last feature is desirable to preventnanoparticles from clumping together due to magnetic attraction. Thecore material may be any magnetizable material such as iron oxide,cobalt, and nickel compounds. Nanoparticles can be non-toxic andbiodegradable if an iron core is used. The core is coated by one or moreshells 114 of non-magnetic material, for example, dextran or silica.Silica coatings are stable and robust, and may avoid the need forrefrigeration. Other polymeric coatings may be considered such aspolystyrene, polyacrylic acid, polyacrylamide and polyvinyl alcohol.

The net field magnitude at location (r,θ) around the nanoparticle hasboth positive and negative variations relative to a uniform field. Thisis shown in the graph of FIG. 2.

While the CPMG procedure refocuses static field non-uniformities, thosewater molecules that move from one field region to another, in the timebetween refocusing pulses, are not refocused and produce T2 effects.Thus, T2 changes are related to the gradient of the net field.

To consider a specific example, the core is Fe₃O₄, with a 4-8 nmdiameter, and the rest of the particle is shell, with an overall 50 nmdiameter. The susceptibility and saturation field depend on thecomposition, crystal structure, and core diameter. Values of thesaturation field range from 0.2 to 0.5 T, and susceptibility ranges from0.2 to 2. A numerical simulation was prepared using 0.5 T saturation and0.5 for susceptibility. The net field in the vicinity of thisnanoparticle is shown in FIG. 2. Strong field enhancements at the two“poles” of the particle are seen, relative to the field reduction aroundthe “equator”. The field within the shell is of no interest and is notcalculated; it is plotted as B₀.

The magnetic field gradient is shown in FIGS. 3 and 4. FIG. 3 is a plotof the magnitude of the field gradient around the nanoparticle. FIG. 4is a plot of the field gradient magnitude along the axis of theparticle. Again, fields inside the particle are not analyzed.

For an echo interval of T_(Echo)=4 msec, the average walk distance isabout 3.5 microns. This is much larger than the length scale of thedistorted-field regions; hence it is safe to assume that the watermolecule has enough time to enter and exit the distorted-field regionbetween refocusing pulses.

The spin dephasing produced by the water molecule passing through thedistorted field region can be estimated as follows. The instantaneousprecession frequency is proportional to the net magnetic field at thewater molecule's location. For simplicity we assume that the moleculerandom-walks through the distorted-field region of one nanoparticle,during one echo interval, starting and ending in the main solvent whichhas only the applied field of B₀. While the molecule is within thedistorted field, it accumulates extra precession compared to the rest ofthe solvent. The phase advance due to the B₀ field is then refocused asusual by the 180 pulses, but the extra precession phase from time spentin the distorted field will not be refocused. The unrefocused phaseincrement due to traversal of a field distortion is the integral of thefield experienced by the particle, minus that in the main solvent:d _(phase)=∫γ(B _(net)(r)−B ₀)dtwhere d_(phase) is the accumulated phase difference between a hydrogenwhich passes through B_(net) (here an explicit function of space) versusone remaining in the uniform field B₀, γ is again the Larmor coefficientand the integral is over the time between refocusing pulses. To obtain arough estimate of the phase shift, the previous equation may besimplified by assuming that the molecule resides in a constant field fora time needed to diffuse through the distorted field region, resultingin the following approximation:d _(phase) =[x _(dis) ²/(2D _(molec))][B _(net) −B ₀]γ

Using the nanoparticle sizes and field assumptions discussed above, thenet magnetic field deviates from the applied field by typically 20 mT.The spins within that field will precess about 850 kHz faster than inthe main solvent. A typical length scale for this distortion isx_(dis)=20 nm. The time needed to diffuse 20 nm is 133 nsec. During thattime, the spins precess an extra d_(phase)=0.1 radians. This representsa substantial dephasing in a single echo interval by a single moleculartraversal, which if not refocused by CPMG will result in a short T2. Inthe sample, many water molecules will be interacting with the nonuniformfield continuously, and each will experience a positive or negativephase shift depending on the specific path. In the ensemble, the extraspin dispersion causes destructive interference and overalldepolarization.

The spin diffusion coefficient in water is in the range ofD_(spin)≈10⁻¹⁵ to 10⁻¹⁶ m²/s, depending on temperature and otherfactors. Although spin diffusion is slower than molecular diffusion, itis sufficient to spread the depolarization among many water molecules ina few msec. Interestingly, solid-state spin diffusion rates tend to bemuch higher, of order 10⁻⁹ m²/s which is comparable to the moleculardiffusion in free water. If the shell exhibits rapid spin diffusion, itcould serve as a conduit for distributing polarization among all of thewater molecules contacting the nanoparticle surface.

Several experiments have demonstrated a T2 increase of 20 to 200 msec.The model suggests that this is due to the analyte molecules obstructingthe surface of the nanoparticle, effectively preventing water moleculesfrom sampling the distorted-field regions at the surface of thenanoparticle.

When analyte molecules attach to the surface of a nanoparticle, aportion of the surface is occluded. The global depolarization rate goesdown and T2 increases. The change in decay rate is roughly proportionalto the fraction of the distorted-field volume occupied by the analyte.If multiple analyte molecules are attached, they all contribute asimilar T2 change on average. If the analyte spends only part of itstime covering up the surface of the nanoparticle, then the T2 changescales proportionately.

A decrease in T2 may also be observed by changing the ratio of thenanoparticles to antibodies. Here antibody is used as an example of theconnection to the analyte. This is defined as stoichiometry control.Depending on the level of detection of analyte one can adjust thestoichiometry to allow rapid detection of analyte.

The reagents and processing conditions may be adjusted to cause adecrease in T2. Formation of extended aggregates of nanoparticles andanalytes is correlated with such a T2 decrease. The model posits thatthe aggregates are open, cage-like structures through which watermolecules may pass easily. This is not explained in earlier studies. Inone embodiment, spin information diffuses in and out of the agglomeratestructure rapidly, so that the depolarization occurring within the cageis equilibrated throughout the sample.

The model suggests that the T2 decrease for agglomerates is due torepeated dephasing when water molecules within the cage repeatedlyencounter depolarizing fields. Such repeated dephasing represents a moreeffective polarization sink than isolated nanoparticles in the freeliquid because the caged water molecule remains in close proximity tonumerous nanoparticle surfaces. While portions of the nanoparticle'sdistorted-field volumes are occluded by analyte, the water moleculecould spend a significant fraction of its time sampling fields thatdiffer from the main field, and thus would become totally dephased in atime short compared to the echo interval. Then, by trading polarizationwith neighboring molecules including those outside the agglomerate, auniformly reduced T2 would result.

The model has utility because it leads to new measurements and new waysof performing measurements related to analyte in the sample. The modelexplains how the analyte interactions with nanoparticles produce bothincreases and decreases in T2, and suggests ways to control the effectsby adjusting reagent concentrations. Noting that speed of detection is acritical parameter for many applications, the model suggests that the T2increase method due to analyte-nanoparticle binding will provide thesignals faster than the T2 decrease from aggregation, because bindingmust occur before the agglomerations. The model also guides thedevelopment of more sensitive nanoparticles using higher-susceptibilitycore material and thinner non-magnetic shells. The model also leads tosteps for canceling systematic errors, such as measuring the T2 of thenanoparticle solution and the sample separately, before mixing, tobetter quantify any T2 changes from the binding. The model also explainshow thermal effects and diffusion effects participate, and can beexploited to accelerate the detection or confirm analyte reactions. Themodel also guides the development of products exploiting the inventivemethods by quantifying signal and noise versus sample size and otherdesign parameters.

Method Description

In one embodiment a method for detecting one or more analytes includes:preparing a liquid sample mixture, which may contain the analyte andother materials; applying a first magnetic field to the liquid;preparing a second and distinct magnetic field within a special regionof the liquid; maintaining the analyte, if any is present, within thespecial region (for example, by providing means for holding the analyte,securing that binding agent adjacent to the special region and allowingthe analyte to interact with the binding agent); exciting magneticresonance signals from the mixture while the analyte is maintainedwithin the special region; analyzing the signals to determine whetheranalyte occupies the special region; and then concluding that analyte ispresent when the signals indicate that the liquid is displaced from thespecial region.

In one embodiment preparing the liquid sample mixture includes the useof a liquid which contains an atom with a nucleus having non-zero spin.The atoms may be an intrinsic part of the liquid, or they may be addedas solute. The step of preparing a liquid sample can include mixing orstirring to ensure that analyte reaches nanoparticles. Mixing can beachieved in numerous ways, including by driving the sample fluidsthrough convoluted tubes using a pump, and such motion may beunidirectional or reciprocal to produce the desired level of mixing.Alternatively, the nanoparticles and the analyte may be contained in thesame type of liquid, so that when the nanoparticles and analyte areplaced in the same container, they spontaneously become mixed withoutthe need for physical stirring. For example, the nanoparticles and thesample material may be dissolved in water and then intermingled bydiffusion in the measurement container. Unassisted mixing may also bearranged by use of highly miscible solvents, such as alcohol and water,for the various ingredients.

The method can also include temperature cycling wherein a sample may beheated or cooled at a fixed location, or the sample may be moved betweenlocations maintained at high or low temperatures. The method can includetaking measurements before, during, and after such temperature changes.For example, a measurement for T2 may be taken immediately upon mixingthe sample, and again after a period of heating and cooling when thesample comes to equilibrium temperature. Comparison of the T2 valuesbefore and after thermal processing will reveal reactions, such asanalyte binding to nanoparticles, which occurred during thermalprocessing.

The method can include the steps of changing the temperature of thesample and then measuring the T2 parameter. Temperature affects thenanoparticle interactions and the magnetic resonance measurement.Selective binding between the analyte and the affinity molecules on thenanoparticles may be accelerated by raising the temperature,particularly for diffusion-limited reactions. Thus the method mayinclude measuring the T2 of a mixture of nanoparticles and unknownswithin the liquid at a first temperature, preferably a sufficiently lowtemperature that the analyte has not reacted with the nanoparticles whenthe measurement is made. The method may then include the step of heatingthe sample to a second temperature sufficient to promoteanalyte-nanoparticle interactions. The method may include measuring theT2 at the second temperature to observe effects of the binding. Themethod may include a further temperature change, such as return to thefirst temperature, and further T2 measurements to confirm that the T2 ofthe sample after the various temperature changes differs from the T2 ofthe sample before the temperature changes. The steps provide manyadvantages, including improved discrimination against interferents,demonstration that the T2 change is due to analyte-specific binding, anda check for instrumental errors.

The method may include heating the sample to a temperature sufficient todisrupt the analyte-nanoparticle aggregations, thus producing a solutionof analyte-nanoparticle binaries, with a corresponding T2 change. Thetemperature may be raised further until the analyte is disbonded fromthe nanoparticles, thus releasing analyte back into the solution andcausing a further T2 change. The temperature may then be lowered untilbinding or aggregation is restored, with corresponding reversion of T2to the earlier value. This behavior in T2 versus temperature wouldstrongly discriminate against interferents or instrumental errors, andwould confirm the presence of analyte.

The method may include the step of measuring the T2 of the samplematerial prior to mixing with nanoparticles. This would reveal a samplematerial which causes a shift in T2, such as a high-viscosity solutionor chelated iron in the sample. When the sample material causes only asmall T2 shift, the measurement may proceed as usual, but in analysisthe T2 of the processed sample may be compared to that initiallyobserved in the raw sample to determine whether analyte is present. Whenthe sample produces a large T2 shift, it may be advantageous to dilutethe sample until its effects are low enough to permit the magneticresonance measurements. Analyte in the diluted sample may then bedetected as described. When the sample produces such a large T2 shiftthat magnetic resonance measurements are prohibited, the invention canflag that sample as un-testable, thereby avoiding a false alarm, or itcan archive the sample for further analysis.

The method can include preparing a magnetic field in a particular way.The field may be prepared by first generating a substantially uniformfirst magnetic field with sufficient intensity to permit magneticresonance measurements, and then perturbing that field locally toproduce a second magnetic field, distinct from the first, within aspecial region. The second field is distinct from the first when themagnetic resonance signals of the liquid outside the special region areinfluenced by or can be distinguished from signals of liquid inside thespecial region. For example, the second field can be created by mixingor dissolving paramagnetic particles, for example, those nanoparticlesdescribe above, in the liquid. The nanoparticles then spontaneouslygenerate the second magnetic field, in a region closely exterior to thenanoparticles, as a result of magnetization of the nanoparticles by thefirst magnetic filed. Alternatively, paramagnetic ions such as chelatediron or gadolinium could be employed instead of nanoparticles. Anadvantage of this approach is that diffusion-limited reaction rates maybe increased due to the higher mobility of metal-ion chelates. Similarions are used in MRI imaging (Gd-DTPA and Gd-DOTA).

Holding the analyte within the special region can be accomplished byreacting or binding or otherwise attracting the analyte to a materialsurface or molecule for which the analyte has particular affinity. Suchholding may be accomplished by hydrogen bonds, ionic forces, covalentbonds, van der Waals forces, electrostatic forces, or any other type ofmolecular or material attachment. For example, the holding mechanism maybe an antibody raised against an analyte protein, or DNA complementaryto analyte DNA sequences and can include any material surface ormolecule for which the analyte has an affinity. Preferably the holdingmechanism also has null affinity or negative affinity for all solutesother than the analyte which may be present. Preferably, the holdingmechanism is secured proximate to the special region, so that theanalyte will be held within the special region. For example, when thespecial region is exterior to a nanoparticle, antibodies to the analyte,or the other holding mechanisms mentioned above, may be attached to thesurface of the nanoparticle, so that the analyte will be held adjacentto the nanoparticle within that region and the liquid will be excluded.Optionally, the nanoparticle may include multiple antibodies, orcomplimentary DNA, or other binding agents so as to interact with anumber of different, but selected, analytes. For example, thenanoparticle could be adorned with complementary DNA for anthrax,antibodies for ricin, and complementary DNA sequences for smallpox,thereby enabling detection of any of these analytes in a single mixture.

The magnetic resonance measurements and analysis to determine whetherthe analyte occupies the special region can include analyzing themagnetic resonance signals by spectral analysis to seek a frequencycomponent characteristic of the special region. That frequencycomponent, if present, indicates that the liquid is in the specialregion, and therefore the analyte is not present. Alternatively the stepcould include applying the CPMG procedure, and analyzing the signals todetermine the T2 of the liquid. The T2 distribution may be a singleexponential component, or it may include a multitude of components,depending on the spin diffusion rate. In either case, however, a T2which is longer than the T2 of the baseline case (liquid with thenanoparticles and no analyte) indicates the presence of the analyte.

A variation of the method includes forming an aggregate comprising aplurality of analyte entities. Then, a reduction in T2 (compared to thebaseline) indicates the presence of the analyte. For example, anaggregate of nanoparticles with attachment mechanisms and analytemolecules may form when both nanoparticles and analyte molecules havemultiple attachment points. Since the aggregation results in a decreasein T2, whereas binding of analyte to nanoparticles results in anincrease in T2, it is important to previously calibrate the signals, sothat the expected sign of T2 change is known in advance. Nanoparticlestoichiometry can be adjusted to prevent agglomeration or to causeagglomeration depending on the measurement process to be used.

In one embodiment, analyte causes nanoparticles to form extendedaggregates. Membrane filters are used to separate those aggregates fromthe liquid medium. The pore size of the filter is preferably larger thanthe size of the nanoparticles or of the analyte, but smaller than theaggregates. When an agglomerated sample is filtered, the filtrate has areduced concentration of both nanoparticles and analyte, which are thusboth greatly concentrated as a filter cake. When secondary analysismeans are desired, for example to confirm detection of a microbe, thefilter cake is used for that secondary analysis. Likewise, the filtrateliquid may be re-measured using the inventive system as an additionalcheck, since the T2 of the filtrate should be much longer than of theinitial nanoparticle solution when most of the nanoparticles have beenfiltered out.

The method may include the steps of measuring the T2 value of astandard. Here a standard is any material which has a known T2.Preferably the T2 of the standard is unchanging in time and is knownfrom prior calibration measurements. For example the standard may be asolution of nanoparticles or of copper sulfate with a concentrationadjusted to provide a particular value of T2. Standards enable detectionand correction of instrumentation drifts. The standard may be a liquidwhich is not a solution, such as an oil selected to have a T2 in thedesired range. The standard may be arranged to have a T2 substantiallyequal to that of an analyte-free sample, in which case it is called anegative comparator. The standard may have a T2 close to that producedby the analyte, a positive comparator. The method may include measuringthe T2 of multiple standards with different T2 values.

The method can include the step of testing a positive and/or a negativecontrol. A positive control can be a benign analyte, such as bacillussubtilis along with nanoparticles sensitized to it. The positive controlmay be analyzed at any time, and should be detected in the same way as athreat analyte. Preferably the T2 change produced by the positivecontrol is known from prior calibration, and testing the positivecontrol should always produce the expected T2 change, and failure to doso would reveal a malfunction in the system. A negative control is abenign analyte along with nanoparticles sensitized to some othermaterials, for example bacillus subtilis combined with nanoparticlessensitized to anthrax. The negative control should never produce a T2change because the analyte and the nanoparticles are not matched. If anegative control produces a T2 change, it would reveal a malfunction ofthe system. An advantage of running positive and negative controls isthat the entire sample collection, fluidics, sample processing, anddetection stages are tested realistically. For comparison, the positiveand negative comparator standards discussed in the previous paragraphtest only the magnetic resonance portion of the system, not the sampleprocessing stages.

The method can include the steps of producing both an increase and adecrease in T2 of the sample. The increase or decrease in T2 depends onthe properties of the nanoparticles, ratios of other reagents such asantibodies, and on other processing parameters. Thus a sample may betested for a T2 increase using processing steps to generate a T2increase when analyte is present, and then the same sample may be testedfor a T2 decrease by adding the ingredients or processing steps whichproduce a T2 decrease. Observation of both increasing and decreasing T2values would enhance the reliability of the analysis and reduce thefalse alarm rate. Alternatively, two aliquots drawn from the same samplemay be processed to generate a T2 increase in one and a decrease in theother.

Interferents are materials which, if present in a sample, cause a changein T2 mimicking that of the target analyte. Most interferents produce ashorter T2, including materials containing chelated iron and materialscausing an increase in viscosity of the liquid. Thus the effects ofanalyte and interferents may be discriminated by processing the sampleso that the analyte will produce a T2 increase. For even greateranalyte-interferent discrimination, both increases and decreases in T2may be arranged, either by sequential processing of the same sample orby comparison of parallel aliquots.

The method can include the step of measuring the T2 of a nanoparticlemixture prior to adding the sample material to that mixture. Theadvantage of this step is that any errors in the nanoparticleconcentration or properties would be revealed before the sample materialis used. If the nanoparticle solution exhibits an unexpected value of T2(for example due to a high or low nanoparticle concentration from ametering error) then the nanoparticle solution may be dumped and a newnanoparticle solution may be prepared. If the nanoparticle solutionexhibits a value of T2 close to that expected, then the nanoparticlesolution may be employed. Preferably the measured value of T2 is thenused in the analysis for comparison against the T2 of the mixed andreacted sample, thereby negating errors due to nanoparticleconcentration and also improving reproducibility.

The method may include the steps of mixing the sample material andnanoparticles in the liquid, then measuring the T2 of the mixture, thenpromoting reactions between analyte and nanoparticles, and thenmeasuring the T2 after such reactions. For example, the sample may beshaken or heated to promote the reactions. Simultaneous mixing andheating may be used to accelerate reactions. Comparison of the T2 of themixture before and after the reactions reveals the analyte. An advantageof these steps is that any errors in the volumes of sample andnanoparticles would be detected and negated.

In one embodiment hazardous chemicals are generally not required. Forexample, analytes can be tested using only water, salts, nanoparticles,and harmless proteinaceous reagents such as antibodies.

System Description

One embodiment of a system which can carry out or implement themeasurement or detection techniques described above will now bedescribed with reference to FIG. 5 which is a functional block diagramof a magnetic resonance system generally indicated as 500. The systemincludes magnet or magnet system 512. In one embodiment the magnet 512is a permanent magnet configured to produce a 0.5 Tesla magnetic fieldwith 0.01% uniformity within a sample area or volume 514 of 1 ml.Alternatively, the magnet system may include an electromagnet, asuperconducting coil, or any other source of magnetic field. A coil orantenna 516 is located adjacent to the sample volume. In one embodimentthe coil encircles the sample volume 514. A pulse generator 518 iscoupled to the coil 516 to provide electromagnetic pulses at the desiredLarmor frequency to the sample volume 514. An amplifier 519 may beplaced between the pulse generator and the antenna to amplify the signalfrom the pulse generator. A receiver 520 is also coupled to the coil 516so as to receive signals picked up by the coil. A preamplifier 521 maybe placed between the receiver and the antenna to amplify the antennasignals. The receiver 520 converts the received signals into a digitalform. A controller 522 is in communication with the pulse generator 518and the receiver 520. The controller controls the operation of receiverand the pulse generator. The controller also receives the signalsreceived by the receiver after they have been converted into the digitalform. The controller 522 can be a general purpose processor, a digitalsignal processor (DSP), an application specific integrated circuit(ASIC), a field programmable gate array (FPGA) or other programmablelogic device, discrete gate or transistor logic, discrete hardwarecomponents, or any combination thereof designed to perform the functionsdescribed herein. Alternatively, the functions of the controller, pulsegenerator, receiver, and user interface may be combined into a singleunit such as an ASIC or FPGA, or a board integrating such circuits. Auser interface system 524 is coupled with the controller 522. The userinterface system 524 provides a mechanism for interaction between a userand the system 500. The interface system can include, for example, adisplay such as a liquid crystal screen, indicator lights, a key board,a mouse, an audio speaker, a microphone, switches, or a touch screen.

The RF coil can be made small enough to interrogate volumes of microliter size sample volumes or large enough to accommodate liters ofsample. This is a scalable system. FIGS. 6a-d are a representation offour configurations of the antenna, each in perspective view. In part aof the figure, a solenoidal coil is shown having a density of windingswhich is constant along the length of the coil. The sample is placedinside the coil for measurement. The coil acts as an antenna to coupleRF energy into the sample nuclei, and also to couple the magneticresonance signal from the nuclei out to the rest of the system.

In part b of the figure, a solenoidal coil having a variable windingdensity is shown. The winding densities are higher at the ends of thecoil than at the middle. An advantage of using a variable windingdensity is that the RF magnetic field generated by the coil may be mademore uniform than that of a coil of the same size with constant windingdensity.

In part c of the figure, a two-turn single-sided coil is shown. Anadvantage of this configuration is that an elongated container such as atube may be inserted and removed without disconnecting either the coilor the tube.

In part d of the figure, a coil configuration is shown wherein fourloops cooperatively generate a transverse RF magnetic field. Elongatedsamples may be inserted without disconnecting the coil or the tube.

The specific user interface and output of the system is highlyapplication-dependent, but will typically include transmission ofinformation dependent on detection of analyte. For example, suchcommunication may involve recording or archiving test results,displaying a threat alert message, illuminating an alarm or beacon, oractivating an acoustical alarm. Communicating data also includes sendingsignals to other devices, such as automatically shutting off an HVACsystem or sequestering a test sample responsive to detection of selectedanalytes. The communication via the user interface can includeelectronic, optical, infrared, radio, microwave, mechanical, oracoustical means, or any other means for transmitting data or commandsresponsive to analyte test results. Additionally, the user interface caninclude remote communication interfaces such as a network interface cardand a wireless access card which are in communication with thecontroller. These can allow an operator or another device to communicatewith the system, to relay commands or retrieve data or convey an alarm.The communication may include transmitting information by the internet,by a local network, or by direct electronic or wireless link.

In one embodiment, the system is configured in two separate chassis, onewith the magnet 512, the pulse generator 518 and the receiver 520. Theother chassis has the controller 522 and the user interface 524. The twochassis exchange information such as commands and data by an electroniccommunication link, for example, cables, a wireless link, or a fiberoptic link. In a preferred embodiment, the communication link comprisesa USB interface employing standard USB connections on each chassis.

The magnetic resonance system 500 can excite magnetic resonance signalsfrom the hydrogen nuclei in water in the sample volume 514 by applyingelectromagnetic pulses for example, radio frequency (RF) pulses,generated by the pulse generator 518 via the coil. The system detectsthe magnetic resonance signals from the hydrogen nuclei in the water byinductively picking up the signals in the coil 516. The receiverprocesses the received signals using amplifiers, mixers, andanalog-to-digital converters.

In one embodiment the system 500 measures the T2 of the water by theCPMG procedure or technique under the control of the controller 522. Themeasurement includes a 90-degree RF pulse generated by the pulsegenerator followed by a 2 msec delay, and then a string of 2000180-degree pulses at 4 msec intervals. The phase of the 180-degreepulses is orthogonal to that of the 90-degree pulse. The proceduregenerates spin echoes in the 4 msec intervals which are received by thereceiver 520. In one embodiment the controller 522 performs an analysisroutine which determines and records the spin echoes, performs FFTanalysis to obtain spectral peaks, finds the maximum value of the peaks,and fits the peak values to a formula with three variables: theamplitude and decay time of an exponential, plus a time-independentbackground. The observed T2 value is the best-fit exponential decaytime.

The analysis performed by the controller includes a comparison betweenthe observed T2 value and a previously calibrated or measured T2 value.The analyte is detected by the system when the observed T2 value of thesample differs from that of an analyte-free sample. The previouslycalibrated T2 value can be determined by measuring a solution of waterwith the same concentration of nanoparticles as is used for themeasurement of the analyte. The T2 of the water is influenced by theconcentration of nanoparticles. The T2 is also influenced by analytebinding to the nanoparticles and occupying the high-gradient regionaround the nanoparticles. In the preferred embodiment, the nanoparticleconcentration is controlled by formulation of the solution. The T2values of the solution without analyte, and with various concentrationsof the analyte, are also known by prior calibration.

In one embodiment the nanoparticles are dissolved or suspended in awater medium. The nanoparticles have a superparamagnetic magnetite corewith a diameter of 8 nm, surrounded by a shell with a diameter of 50 nm.Antibody molecules (or other binding or attracting mechanism asdescribed herein) specific to the analyte are bound to the shell. Whenthe nanoparticles are in the sample 514, the core is magnetized by thefield applied by the magnet 512, producing a local dipole field whichadds to the applied field. The resulting net field includes spatialgradients of up to 0.1 T/nm, within a region extending radially from thesurface of the nanoparticle to about 20 nm from the surface. Thenanoparticles are most effective for detection and measurement purposesin low concentrations of about 1:10000 in water. That results in verylittle consumption of the nanoparticles per test. In one embodiment themagnet 512 of the magnetic resonance system 500 uses a permanent magnetfor this purpose. The permanent magnet requires no power, may be madearbitrarily compact, and is economical. Most prior magnetic resonancesystems employed electromagnets or superconducting coils to generate themagnetic field. It is not feasible to arbitrarily reduce the size ofelectromagnets or superconducting magnets. If an electromagnet is scaleddown in size, the magnetic field scales proportionately. If the field isheld constant, then the current density in the electromagnet coils mustbe increased. Current density can not be increased arbitrarily becauseof a fundamental limit, the conductivity of copper. Above a certaincurrent density limit, roughly 100 amps/cm², the coils must bewater-cooled. Above a second limit, roughly 200 amps/cm², the coilsself-destruct. Small, high-field, steady-state copper coils are notfeasible.

It is likewise not feasible to reduce the size of superconductingmagnets arbitrarily. Superconducting coils may be made much smaller andmore powerful than nonsuperconducting coils, and can carry high currentdensities. However, superconducting coils must be surrounded by avacuum-insulated cryostat, usually having multiple shells maintained atdifferent cryogenic temperatures. Also, the various shells aremechanically and thermally interconnected by support struts. It is notpossible to make the cryostat arbitrarily thin because of the thermalconductivity of support members. The cryostat limits the miniaturizationfeasible in superconducting magnets.

Permanent magnets have neither of these defects. A given magnet designusing permanent magnets will scale precisely, with no change in geometryor field or field quality, to arbitrarily large or small dimensions. Theonly fundamental limitation is the ferromagnetic domain size, about 1micron. By designing permanent magnet systems, the magnets may be scaledto a size determined by the sample volume, the RF coil properties, orother parameters of the system, rather than forcing the other parametersto comply with the magnet scale. As a result, it is feasible tominiaturize the entire electromagnetic system. This leads to improveddetection sensitivity in smaller sample volumes, reduced cost and weightof the sensor portion of the system, and reduced RF power required.

One embodiment of the magnet 512 is depicted schematically in crosssection in FIG. 7. The magnet includes a frame 710, such as a hollowsteel frame. In one embodiment, the height H of the frame is less than50 cm and may be less than 5 cm. The width W can also be less than 50 cmand can be less than 5 cm. An upper permanent disk magnet 714 and alower permanent disk magnet 716 located opposite the upper permanentmagnet are attached to an upper section of the frame and a lower sectionof the frame, respectively. For example, they can be mechanicallyattached using screws or bolts and/or they can be attached with anadhesive. A disk shaped upper pole piece 718 is located atop the upperpermanent magnet and opposite a disk shaped lower pole piece 720 locatedatop the lower pole piece. Around the periphery of each pole piece areeight fine-threaded holes with adjustment bolts, which may be varied toimprove the uniformity of the field. The magnet is assembled by boltingthe frame together, sliding the permanent magnet disks into position,sliding the pole pieces into position, and then shimming. The permanentmagnet disks are very strongly attracted to the steel frame, and thepole pieces are very strongly attracted to the permanent magnet disks.The attractions, and resulting friction among the various contactingmembers, provide mechanical stability to hold the assembly together.Further robustness may be obtained by applying clamps or adhesives tothe magnet disks or pole pieces, preferably not interfering with fieldshimming or magnetic resonance measurements. Forces on permanent magnetcomponents are strong and potentially dangerous. Not shown are jigs andtools used to control the assembly process in view of the strong forcesinvolved.

Shimming is the process of adjusting a magnet, such as magnet 512, toproduce the necessary uniformity. As built, most magnets provideinsufficient uniformity due to manufacturing tolerances. Shimmingconsists of measuring the field distribution, adjusting built-inparameters of the magnet, and repeating until the desired uniformity isachieved. In one embodiment a simple shimming design is utilized whichfocuses on the most important field parameters, rather than providing anexhaustive set of parameters of which most are never needed.

First, the magnetization of the two permanent magnet disks is equalized.Based on the observed axial gradient, one or more thin ferromagneticsheets are affixed by magnetic attraction circumferentially around onlythe stronger of the two magnets. Iterative adjustment of the number andthickness of the sheets results in near-perfect negation of the axialgradient. The sheets may then be secured by clamps or adhesives.

Then, one or more of the miniature bolts, for example bolt 722, in theperiphery of the pole pieces are adjusted. These bolts press against thepermanent magnet disks to slightly rock the pole pieces as needed tonegate transverse field gradients. Either or both pole pieces may beadjusted, depending on the details of the observed field. Finaladjustment of the various bolts results in near-perfect negation oftransverse gradients.

Typically the shape of the pole pieces need not be altered, althoughthey can be demounted and their shape revised if needed to achieve thedesired field. Alternatively, the spacing between the pole pieces may bereduced slightly by tightening all of the bolts around both pole pieces.Such an adjustment is almost equivalent, magnetically, to adjusting thedepth of the pole piece relief step.

To fabricate the magnet parts, powdered metals such as iron or steel canbe placed inside a mold of desired shape. Then in the press pressure andheat are applied to generate the final part. While only small parts canbe made by this technique, mass manufacturing can be achieved.Alternatively machining can be used to make the individual parts.

The pole pieces can be designed to provide the highest field uniformityand field volume for sample testing, with the constraint that the gap besufficient for inserting and tuning the magnetic resonance sample coil.Design constraints include the maximum field in the pole pieces to limitsaturation, minimum number of shimming parameters to achieve targetfield uniformity, and use of low-cost commercial permanent magnetcomponents where possible.

The permanent magnet material provides very high magnetization density,but is temperature sensitive. In applications where the frequency may beadjusted to the field, thermal drift of the magnetic field is not aproblem. For precision T2 measurements, however, it is necessary tostabilize the magnetic field. A temperature-controlled enclosure can beused. In one embodiment, the enclosure can be built using foaminsulation and a pair of patch heaters. A thermocouple sensor andcontroller complete the arrangement.

Precise determination of T2 using the CPMG procedure is enhanced with anextremely stable local oscillator with minimal phase noise on a timescale of at least the spin echo spacing. Even high-cost crystaloscillators usually do not provide sufficient stability due to the noisycomputer power lines. Sufficient stability can be obtained usinginexpensive integrated crystal oscillators by buffering both the DCpower input, and the RF clock output. Such an arrangement is depictedschematically in FIG. 8. In one embodiment the oscillator shown in FIG.8 is used in the pulse generator 518 of FIG. 5. In general, the DC(direct current) power input is buffered by wiring two or more voltageregulators in series. The circuit depicted in FIG. 8 includes a firstvoltage regulator 802 (for example an 8 volt regulator which receives a+12V input). A second voltage regulator 804 receives the output of thefirst voltage regulator and provides its output to the oscillator 806(for example, a 5 volt regulator, receiving the output of the 8 voltregulator). A third voltage regulator 808 (for example, a 5 voltregulator) can also receive the output of the first voltage regulatorand can provide its output to a digital logic gate 810 with high speedand high source isolation, such as the 74F3037 line driver NAND(available from Philips Semiconductors and others). The digital logicgate 810 buffers the output of the oscillator.

The magnetic resonance system 500 (FIG. 5) interacts with the sampleusing the antenna 516 which, in operation, is electromagneticallycoupled to the precessing nuclei of the sample. In one embodiment thecoil is mounted in a modular, interchangeable platform to enablechanging the sample size, replacing the coil in case of contamination,or other changes needed.

The antenna may be encapsulated in a contamination-resistant material.Contamination is a serious issue when multiple samples bearing multiplediseases or toxins are to be tested. Prior antennas are difficult toclean because they are highly convoluted geometrically and includenon-hygienic insulator and conductor materials. Encapsulation of theantenna can resolve this issue. For example, the antenna could be acopper coil embedded in a hollow cylindrical TEFLON form so that anycontamination coming from the sample container would encounter only aTEFLON surface, never the actual conductor. Since TEFLON isnon-absorbing and relatively easy to clean up, contamination issues aregreatly reduced. Also, the encapsulated antenna would be more stable andmechanically rugged than a freely mounted coil. Magnetic resonancesignals from an element in the encapsulant, such as deuterium orfluorine, may be used to control a frequency or a magnetic field.

Cancellation of noise, interference signals, baseline offsets and otherbackground effects can be improved by performing magnetic resonancemeasurements multiple times with various RF phases alternated. This canbe implemented under the control of the controller. For example, theexcitation may be alternated between positive and negative phaserotation of the spins during RF pulses. During signal processing by thecontroller, the phase of the receiver oscillator can also be rotated by90 degrees or its multiple. Analysis software in the controllercontrolling these phase alternations also performs the correspondingaddition or subtraction of the digitized data to accumulate the desiredsignal while canceling backgrounds.

Various user interfaces can be provided with the system. For example,the system 500 depicted in FIG. 5 can carry out measurements to detect aselected analyte or analytes and report the results by issuing an alarmif detected or provide a visual indication or report via the userinterface 524. In one version, the operator inserts a mixed sample intothe system and presses a single button on the user interface to initiatea previously prepared series of instructions for the controller to carryout and analyze the sample. If more than one analyte is to be searchedfor, the instructions automatically direct the mixing of nanoparticlessensitized to each analyte and carries out the measurementssequentially. In another version of the instrument, a mechanical oroptical switch senses the insertion of the sample into the magneticresonance system, and automatically initiates the measurement sequence.

In one embodiment, a T2 change is the primary indicator that analyte ispresent. To check for drifts or errors which could affect the T2measurement, the system can compare the measured T2 of the sample, withthat of a sealed calibration sample having a previously measured T2value. The sealed sample may contain copper sulfate in water, mineraloil, or other liquid having a stable T2 for comparison. Alternatively,the sealed calibration sample can be periodically measured.

A wide diversity of mechanisms for presenting the sample into themagnetic resonance system can be used. The sample, comprising liquidmedium, analyte, and nanoparticles, can be mixed in a container such asa glass NMR tube, a plastic tube or vial, a disposable container such asa plastic Eppendorf tube or flask, or other suitable container. Anadvantageous polymer is PEEK (polyetheretherketone) due to itstoughness, inertness, and machinability. The container may be coatedwith a material to prevent nanoparticles from adhering to the walls,clumping, or precipitating out of the mixture. For example, the coatingmay be a protein such as BSA (bovine serum albumin). The containerincluding the sample may be inserted, manually or by a mechanicalfeeder, into the magnetic resonance system. Alternatively, a fixedcontainer in the magnetic resonance system may be used for multiplesample measurements by inserting sample liquids into the container, forexample by pumping the sample or its ingredients through tubes into thecontainer. After the measurements, the sample is then drawn from thefixed container using pumps, tubes, valves, and related fluid flowdevices. A washing or rinsing step can be carried out between samples.Ultraviolet treatment of reservoirs holding distilled water andnanoparticles can be carried out to prevent bacteria formation.Alternatively, a fungicide such as sodium azide can be mixed in thedistilled water in trace quantities to prevent growth of bacteria andalgae in the water.

In one embodiment depicted schematically in FIG. 9, multiple sensorunits are connected to a single controller. For example, an automated,fixed-site system may consist of one central controller 902 with powersupplies and a pulse generator or transmitter, connected by cables tomultiple remote sensor heads 906 a and b. Though only two sensor headsare depicted, more can be used. Each head 906 includes a samplepreparation apparatus along with selected nanoparticles, a magneticresonance magnet, a preamplifier and a coil, for example as weredescribed in connection with FIG. 5. RF power pulses are routed to thesensor units through an output multiplexer 908 which is controlled bythe controller 902. Signals from the sensor units are routed to thereceiver 910 through the input multiplexer 912, also controlled by thecontroller. Interconnects are preferably by coaxial cable.Alternatively, each sensor unit may include an RF amplifier. When the RFamplifier is located at the sensor unit, the interconnects do not carryhigh power RF pulses and thus may be wireless, fiber optics, or othercommunication means as well as coaxial cable. The elements of the systemdepicted in FIG. 9 operate in the manner described above.

In one embodiment, particulate matter suspended in air may be drawn fromfree air, HVAC ducts, interior spaces such as shopping malls, subwaytrains and other mass transit areas, or any other air system to test fordiseases or terrorist attack. (HVAC stands for heating, ventilation, andair conditioning.) Collection preferably includes drawing particulatematter into the system or concentrating particles from the air into theliquid medium. FIG. 10 shows a schematic of such a monitor system. Thecollector 1002 can be situated within a duct or in any other area to bemonitored, and can include a shroud (not shown) to exclude dirt andinsects. The collector 1002 can include an electrostatic concentrator toattract analyte or sample material. A fluidics system 1004 transportsthe analyte from the collector 1002 to the sample area of the magneticresonance analyzer or system 1006. The magnetic resonance system 1006can be the system described in connection with FIG. 5. The fluidicssystem 1004 can include an automated microfluidic mixer to mix analytewith a liquid, such as the water medium and with nanoparticlesconfigured for the one or more analytes to be detected. A reservoir ofthe nanoparticles and the water 1008 can also be part of the fluidicssystem. The mixed sample is then transferred by the fluidics system tothe sample area of the magnetic resonance system where measurements aremade. In one embodiment a fluidic transport system is in communicationwith the mixer and extends into the sample area. Depending on themeasurement results, the sample may be dumped into a waste container,stored as archive material, or sent to secondary analysis systems. Thewaste water may be recycled to be used again by passing through afilter.

FIGS. 11a-e depict an embodiment of a fixed installation system asdescribed in connection with FIG. 10 and three collector intakes. FIG.11a is a perspective view and FIG. 11b is a plan view of the systemshowing the intake 1102 and a display 1104. The other elements depictedin FIG. 10 are contained within the casing. FIGS. 11 c-e depict threeinlet options for the system. Once started the controller causes thesystem to collect samples periodically for analysis. The system can alsobe operated manually. A user interface may be provided through buttonsor a touch screen. The display 1104 can show the status of operation.User access can be controlled through, for example, biometricidentification, such as fingerprint identification, or a password.

FIG. 12 is a front perspective view of a hand portable system. Thesystem can operate in a single button autonomous operation mode. Asample can be introduced via vials and tubes. A sample in a container1202 can be introduced into the system through a receptacle or opening1204 at the top. Inside the system the fluidics system will handle thesample mixing and moving into the NMR system in the manner describedabove in connection with FIG. 10. The user interface 1206 can include“biohazard” and “safe” lighted areas on a display screen. To startoperation, a start button is provided on the touch screen. The status ofsystem operation is indicated on the screen.

One embodiment of a system which can carry out or implement themeasurement or detection techniques described above for medicaldiagnostic purposes will now be described with reference to FIG. 13which is a functional block diagram of an automated sample testingsystem. For clinical applications, the sample comprises a specimen ofmaterial from a patient. The material may include living or deadcellular material such as skin, blood, prions, marrow, hair, biopsysamples, or other tissue; or non-cellular biological material such assaliva, mucous, sputum, intravenous fluid, urine, feces, pus, spinalfluid, and contents of the stomach or intestines; or any other samplematerial obtained from a human or animal body. Collecting that materialcomprises a patient or subject producing the material, a clinicianextracting the material from the body of a patient or subject, aninvestigator retrieving sample material from a crime scene or accident,or any other steps resulting in the accumulation of biological materialfor testing.

First, the fluidic system 1302 draws a patient's specimen 1304, or aportion thereof, or a solution thereof, into a mixer which mixes thesample material with a solvent, for example stored in a solventreservoir 1306, and one or more types of nanoparticles stored inreservoirs 1308 a-c. Each type of nanoparticle can be sensitized for oneor more chemicals or analytes related to diseases or medical conditions.FIG. 13 shows three nanoparticle types, but any number of nanoparticletypes, each sensitized to one or more analytes related to one or moremedical conditions can be used. Diseases include communicable pathogenssuch as viruses and bacteria, and non-communicable diseases such ascancer or hypercholesteremia. Chemicals include enzymes or other markersproduced by the body, toxins, and drugs. In one embodiment, the userselects the types of nanoparticles to be used in testing a particularpatient's specimen. For example, a physician may extract a sample of apatient's blood to check the concentration of a medication so as tocontrol dosage, or guards at an airport or border crossing may taketissue samples of live or dead chickens to check for avian flu.Additional processing steps may include lysing the sample to release DNAor RNA or other components of the sample, heating or cooling the sample,adjusting the pH of the sample, or other steps needed to promoteselective reaction between the nanoparticles and the analyte. The mixedsample, or an aliquot thereof, is then transferred into the magneticresonance system 1310, such as the system depicted in FIG. 5. The samplemay alternatively be mixed with nanoparticles within a container whichis within the magnetic resonance instrument, thereby avoiding the stepof transferring the mixed sample, and additional processing steps may betaken while the sample is within the magnetic resonance instrument.

The magnetic resonance instrument then measures signals from the sample,such as the T2 of the sample, and analyzes those signals to determinethe presence or absence or concentration of the selected analytes. Then,based on the measurement results, a physician may then diagnose thepatient's disease.

In one embodiment of the systems described above, the system detectsanalyte by measuring signals from the liquid, the signals being relatedto the magnetic field. Specifically, the signals are sensitive to thedistinct magnetic field in the special region around the nanoparticles.When analyte binds to the corresponding antibody or other binding agent,the analyte is caused to remain in the special region, and thus in thedistinct magnetic field. The analyte displaces the liquid from thatregion, so the liquid no longer emits magnetic resonance signalscharacteristic of the magnetic field in that region. Also, it isimportant to note that the analyte does not emit magnetic resonancesignals, or at least does not emit signals which are similar to those ofthe liquid. This is because the analyte is held tightly to the solidnanoparticle, causing the analyte to exhibit the short T2 characteristicof solids. Thus, in one embodiment, the analyte, while occupying thespecial region, does not produce signals that mimic the liquid.

Agglomeration can cause a change in T2 but not T1, whereas both T1 andT2 change in response to increased concentration of nanoparticles.Therefore, a measurement of T1 can be used as a calibration or anindependent measure of nanoparticle concentration. In one embodiment,the system measures both the T1 and T2 of the sample, applies analysisrelating the T1 value to determine the nanoparticle concentration, andthe T2 value to detect analyte. Alternatively, other methods areavailable to measure the iron content, and hence nanoparticleconcentration, in the sample.

The data processing step performed by the controller includes fittingthe data for parameters related to the presence of analyte, such as a T2change in CPMG data. Normally the echo train in CPMG is fit to a singleexponential formula, a three-parameter fit for amplitude, time constant,and background. A simple but efficient way to accomplish this is a gridsearch in which all three parameters are first estimated from the data,and then a three-dimensional grid of values is generated by varying allthree parameters above and below the estimated values. Then the bestvalues are selected as the minimum chi-square, or mean squared deviationof the data from the formula. Starting from the best value, a new searchgrid is again calculated, the deviations calculated, and the best valuesagain derived. This process is repeated a number of times (typically 9)to obtain the best global fit. Optionally, the scale of the grid may bereduced by a factor (typically 0.95) each time it is used, so that thesame values are not appearing repeatedly.

The primary subsystems of the magnetic resonance system are the pulsegenerator, the signal receiver, and the controller. These subsystems mayreside on separate boards, interconnected by cables. Alternatively, thesubsystems may be integrated as a single circuit on a single computerboard. The advantage of the latter is that cable interconnects are notneeded, and also that a single time base may be used for all.

The system can be battery powered. The system uses very little powerduring data acquisition, and can be programmed to use essentially zeropower in a sleep mode.

In one embodiment the system also includes a radiation detectorinterfaced to the controller. The purpose of the radiation detector isto detect radioactive materials in the sample. The radiation detectormay be any radiation sensor, preferably sensitive to gamma rays, such assemiconductor, scintillator, and gas-filled counters. The detector maybe positioned proximate to the sample collection means, the samplemixing system, or a holding chamber placed downstream of the magneticresonance system.

Insects such as spiders may obscure the air inlets and collectors. Firstbarrier to entry for these bugs are filters. For outside installations,a slow release insecticide, preferably harmless to humans and pets, canbe incorporated. Such insecticides can be implemented along the shaft ofthe inlet or near the mouth of the inlet.

In one embodiment the systems and methods detect explosives and chemicalweapon materials. The systems and methods can perform the detectionusing nanoparticles as disclosed above, wherein specific binding siteson the nanoparticles bind to the explosive or chemical weapon molecules.Alternatively, the systems and methods can detect explosives or chemicalweapon materials by measuring magnetic resonance signals from the samplematerial itself, without use of nanoparticles. The system may employ theSpin Nuclear Overhauser Effect to detect chemical weapons andexplosives. No nanoswitches are required in this case. Anotherconfiguration could be a hybrid system incorporating gas chromatography,mass spectroscopy, ion mobility spectroscopy, other analyticaltechniques, and NMR with or without nanoparticles

An advantage of the inventive systems and methods is that confirmationtests may be carried out for certain analytes using the same apparatus.For example, a confirming test for explosives comprises measuring the T1parameter using a magnetic resonance system, since the T1 for mostexplosives is extremely long (many seconds). As another example, aconfirming measurement for chemical weapons such as nerve agents is amagnetic resonance scan for fluorine or phosphorus based on thecharacteristic Larmor frequencies of those elements.

In one embodiment the system detects toxins and biological weapons inmail envelopes, by testing particulate matter collected from mail. Inthis application, the system would preferably include means forextracting particulate matter from envelopes, such as shaking,vibrating, blowing air through the mail piece or compressing theenvelopes. The system may include means for cutting envelopes toretrieve powder, preferably only after other sensors had directedsuspicion at a particular mail piece.

A preferred embodiment for applications sampling air includes an airinlet, a collector, concentrator and an automated fluidic system. Theair inlet includes a filter to exclude dirt and insects, and a cycloneto separate sample particles from air. Inlets may use “impactor” or“pre-separator”, or “fractionator” and serves the role of preventinglarge (e.g., particles with sizes greater than about 10 micrometersaerodynamic diameter) from entering the detector or identifier. Thelarge-particle fractionator is an integral component in the ambientsampler—it is the combination of the internal nozzle and the plate thatis normal to the nozzle. For the HVAC unit or the occupied environmentsampler, there could be an optional pre-separator cartridge that isplaced downstream of the inlet. In addition, for the ambient sampler,there could be a bug screen that is placed just upstream of the exhaustport. The collector includes concentrator means including a virtualimpactor to insert the sample particles into a liquid medium. Thefluidic system then mixes the sample with nanoparticles.

In one embodiment the systems and methods are adapted to inspectshipping containers, for example to detect hazardous materials or drugsor microbes among items in a shipping container. The embodiment includesmeans for drawing air from the interior space of the shipping container,means for collecting or concentrating any material suspended orentrained in that air, means for mixing the material with nanoparticles,and means for presenting that mixture to the magnetic resonance systemfor testing. The inspection may be carried out by opening a door of theshipping container. Alternatively, the interior air may be drawn througha port or reclosable opening on the shipping container. Further detailsare provided in Provisional Application Ser. No. 60/669,019, filed Apr.7, 2005, titled SHIPPING CONTAINER INSPECTION DEVICE.

Those of skill will further appreciate that the various illustrativelogical blocks, modules, circuits, and algorithm steps described inconnection with the embodiments disclosed herein can often beimplemented as electronic hardware, computer software, or combinationsof both. To clearly illustrate this interchangeability of hardware andsoftware, various illustrative components, blocks, modules, circuits,and steps have been described above generally in terms of theirfunctionality. Whether such functionality is implemented as hardware orsoftware depends upon the particular application and design constraintsimposed on the overall system. Skilled persons can implement thedescribed functionality in varying ways for each particular application,but such implementation decisions should not be interpreted as causing adeparture from the scope of the invention. In addition, the grouping offunctions within a module, block, circuit or step is for ease ofdescription. Specific functions or steps can be moved from one module,block or circuit without departing from the invention.

The various illustrative logical blocks, modules, and circuits describedin connection with the embodiments disclosed herein can be implementedor performed with a general purpose processor, a digital signalprocessor (DSP), an application specific integrated circuit (ASIC), afield programmable gate array (FPGA) or other programmable logic device,discrete gate or transistor logic, discrete hardware components, or anycombination thereof designed to perform the functions described herein.A general-purpose processor can be a microprocessor, but in thealternative, the processor can be any processor, controller,microcontroller, or state machine. A processor can also be implementedas a combination of computing devices, for example, a combination of aDSP and a microprocessor, a plurality of microprocessors, one or moremicroprocessors in conjunction with a DSP core, or any other suchconfiguration.

The steps of a method or algorithm described in connection with theembodiments disclosed herein can be embodied directly in hardware, in asoftware module executed by a processor, or in a combination of the two.A software module can reside in RAM memory, flash memory, ROM memory,EPROM memory, EEPROM memory, registers, hard disk, a removable disk, aCD-ROM, or any other form of storage medium. An exemplary storage mediumcan be coupled to the processor such the processor can read informationfrom, and write information to, the storage medium. In the alternative,the storage medium can be integral to the processor. The processor andthe storage medium can reside in an ASIC.

The above description of the disclosed embodiments is provided to enableany person skilled in the art to make or use the invention. Variousmodifications to these embodiments will be readily apparent to thoseskilled in the art, and the generic principles defined herein can beapplied to other embodiments without departing from the spirit or scopeof the invention. Thus, the invention is not intended to be limited tothe embodiments shown herein but is to be accorded the widest scopeconsistent with the principles and novel features disclosed herein.

What is claimed is:
 1. A method for detecting whether an analyte ispresent using magnetic resonance and paramagnetic particles, the methodcomprising: applying a first magnetic field to a sample comprising amaterial to be analyzed, a known liquid and paramagnetic particleshaving an affinity material, wherein the affinity material binds theanalyte but does not form agglomerations; applying a second magneticfield within at least one region of the sample such that the magneticresonance signals of the liquid in the at least one region are differentfrom magnetic resonance signals of the liquid exterior to the at leastone region, wherein the second magnetic field is created by theparamagnetic particles in the presence of the first magnetic field;holding the analyte with the affinity material in the at least oneregion, so as to inhibit spin-spin relaxation in the known liquid,thereby causing an increase in the spin-spin relaxation time (“T2”) ofthe known liquid; exciting magnetic resonance signals from the liquidwhile the analyte is in the at least one region; determining the T2 ofthe sample from the magnetic resonance; and determining the presence ofthe analyte by determining whether the determined T2 is greater than theT2 of the combination of the known liquid with the paramagneticparticles.
 2. The method of claim 1 wherein the second magnetic field issuch that magnetic resonance signals of the known liquid in the secondmagnetic field can be distinguished from magnetic resonance signals ofthe liquid exterior to the second magnetic field.
 3. The method of claim1 further comprising determining the amount of the analyte in the samplefrom the determined T2.
 4. The method of claim 1 wherein thestoichiometry of the paramagnetic particles prevents agglomeration. 5.The method of claim 1 further comprising measuring T2 of the liquid andparamagnetic particles without the material to be analyzed.
 6. Themethod of claim 1 further comprising measuring the longitudinalrelaxation time (“T1”) of the sample to determine the concentration ofnanoparticles in the sample and using that determination to determine abaseline for determining whether an increase in T2 has occurred.